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Journal of Natural Products Vol. 58, NO.8,pp. 1261-1264, August 1995
STRUCTURE/ACTIVITY RELATIONSHIP OF SOME NATURAL MONOTERPENES AS ACARICIDES AGAINST PSOROPTES CUNICULI STEFANIA PERRUCCI,*
G U I DMACCHIONI, ~
Dipartimento di Patologia Animale Proflassi ed lgiene degli Alimenti, Universitlj di Pisa, Viale delle Piagge 2, 56124 Pisa, Italy
PIERLUIGICIONI,G u m FWI,
and Ivmo M~RELLI
Dipartimento di Chimica Bimganica, Uniwsitlj di Pisa, Via Bonanno 33, 56126 Pisa, Italy AssTMcr.-The pharmacological activity of many essential oils on a large number of human and animal pathogens, as used in folk medicine, has been confirmed world-wide by several laboratory investigations. Unfortunately, the biological properties of essential oils can be extremely inconsistent because of the variability of their chemical composition. The acaricidal activities of some natural terpenoids, which are the main constituents ofseveral essential oils, were evaluated in vitro against the mange mite (Psoroptescuniculi)of the rabbit , by direct contact and by inhalation. Because the test components represent different chemical classes (hydrocarbons, alcohols, and phenols, with free and esterified or etherified functional groups), it was also possible to discern in a preliminary fashion a correlation between chemical structure and acaricidal activity. The results obtained suggest that molecules possessing free alcoholic or phenolic groups showed the most potent acaricidal activity.
The biological activity of essential oils toward several microorganisms (mainly bacteria and fungi), but also toward arthropods, has been confirmed by many reports (1-7). Data also show great inconsistency, however, among these materials; the reasons for the variability of their composition are due to many environmental (i.e., climate, soil type, height above sea level, etc.) and genetic factors influencing the biosynthetic pathways of the secondary metabolites. Therefore, the identification of the main constituents responsible for the activity of a crude essential oil is of interest. A comparative study of the activity of each compound, even if it does not permit assessment of the potential synergy and antagonism among the components of an essential oil, could enable a determination of the necessary structures for their pharmacological action. This information should also allow the prediction of the biological activity of other structurally related chemical substances, and the assessment of their possible modes of action. In the present study we have evaluated in vitro miticidal properties against
Psoroptes cuniculi (Delafond, 1859), by direct contact and by inhalation, of some monoterpenes which are the main constituents of several essential oils. In order to ascertain the possible structure-activity relationships, simple hydrocarbons (limonene, myrcene, y-terpinene), alcohols (linalool, geraniol, nerol, terpinen-4-01, a-terpineol, menthol),and phenols (thymol, eugenol), with free ester (linalyl acetate) or ether (estragole) functional groups and open or closed chains, were chosen for testing. In a few cases, namely nerol and geraniol, the effect of stereochemistry of the molecules was investigated in order to further define the structure-activity relationships. Although other investigators have attempted to define the main components responsible for the antibacterial, antifungal, and insecticidal activity of essential oils (7-14), there appear to have been no studies to date that examine systematically the acaricidal activity of these substances with reference to their chemical structures. The results obtained are summarized in Tables 1 and 2. Table 1 shows the lethality of the test compounds and Table
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0701. 58, No. 8
Journal of Natural Products
TABLE 1. Acaricidal Activity (Percent Lethality) of Terpenes Against Psoroptes cuniculi. Mean Values of Six Redications2Standard Deviation. Direct Contact
Inhalation
Compound Limonene . . . . . . . Myrcene . . . . . . . .
1.0%
0.25%
0.125%
6.0 p1
3.0 p1
1.0 pl
0.0
0.0 0.0
0.0
0.0
0.0
6.72
0.0 0.0 5.2 0.0 75.0t 8.4 93.32 8.2 83.32 10.3 41.72 11.7 98.32 4.1 100.0 13.32 5.2 100.0 6.7 2 8.2 96.7 2 5.2
0.0 0.0
0.0
y-Terpinene . . . . . Linalool . . . . . . . .
100.0
0.0 100.0
Geraniol . . . . . . . .
100.0
100.0
100.0
100.0
96.7 2 5.2 100.0
100.0
100.0 98.32 4.1 100.0 20.0? 20.0 100.0 5.02 8.4 100.0
Nerol
........
0.0
0.0 97 .O2 5.2 100.0
Terpinen-4-ol. . . .
100.0
98.32 4.1 100.0
. . ...
100.0
100.0
100.0
100.0 40.02 12.6 100.0 100.0
100.0 35.02 12.2 100.0 63.32 10.3 100.0
100.0 5.02 8.4 100.0 0.0
a-Terpineol
Menthol' . . . . . . . Linalyl acetate . . . Thymol' . . . . . . . . Estragole . . . . . . . Eugenol
.. . .. . . .
100.0
100.0
0.0 100.0
0.0 36.72 8.2 95.02 5.5 36.72 10.3 5.02 8.4 73.32 5.2 100.0 0.0
100.0 0.0 86.72 8.2
'Substances, in inhalation assays, diluted 1:6 (w/w) in vaseline oil and used at 36, 18,6 p1.
2 the lethality percentage of the controls. In the direct contact tests all monoterpene hydrocarbons, either alicyclic (i.e., myrcene) or cyclic (i.e., limonene and yterpinene) did not show any miticidal activity at the doses tested. The doublebond position and/or number seems to be unimportant for this kind of biological activity. Incontrast, the terpenealcohols, such as linalool, geraniol, nerol, menthol, terpinen-4-01, and a-terpineol, are able to kill by direct contact nearly 100% of
Compound Acacedenh . . . . . . . . . . Vaseline oil . . . . . . . . . . . Physiological saline . . . . . Untreated controls . . . . . .
mites at the doses tested. Therefore, the the oxygenated functional groups potentiate the acaricidalproperties among these compounds. Neither the alicyclic (i.e., linalool, geraniol, nerol) nor cyclic (i.e., menthol, terpinen-4-01, a-terpineol) nature of the compound appeared to influence miticidal activity. Similarly, neither the sites of linkage to the ring or to a side-chain, nor the nature of the hydroxyl group (primary, secondary, or tertiary), influenced the activity. The cis/ trans isomerism represented by nerol and
Direct Contact 100.0
5.028.4 1.7 24.1 8.327.5
Inhalation 0.0 6.725.2 5.025.5 9.720.8
'Acacerulen" active principles: 1% dichlorophen and 1% of a pyrethrum extract containing 0.25% of pyrethrins.
August 19951 Perrucci et al. : Miticidal Activity of Monoterpenes geraniol also seems to be unimportant. Thymol and eugenol killed nearly 100% of the parasites at all dosages used in the direct contact tests, indicating that aphenolic function can enhance the miticidal characteristics of terpenes. The low susceptibility of mites to linalyl acetate, particularly at the lowest doses, could be related to the esterification of the oxygenated function. Estragole, structurally close to eugenol, but with a methylated phenolic function exhibited, at a concentration of 1%, an activity comparablewith the same dose of eugenol, but at 0.25% this action decreased (63%) and fell to zero at 0.125%. These results indicate that the best miticidal activity of the monoterpenes examined, in direct contact tests, can be related to compounds with free alcoholic or phenolic functional groups. Experimentswith themites subjected to inhalation of the volatile fraction showed that, at 6 p,l, the active compounds demonstrated results comparable to the direct contact tests; thus, while simple hydrocarbons were ineffective, alcohols and phenols maintained almost 100% miticidal activity. Lowering the dose to 3 PI, all the alcohols represented possessed nearly the same acaricidal action, except nerol (83.3%) and terpinen4-01(41.7%). At 1 p,l,geraniol,menthol, and thymol maintained their acaricidal properties unchanged, while the activity oflinalool, eugenol,a-terpineol, terpinen4-01, and nerol was diminished. Linalyl acetate and estragole, like the hydrocarbons, were partially or completely ineffective at all doses tested. The identification of novel active natural compounds could increase the number of available chemotherapeutic agents, thereby reducing the frequencyof resistance phenomena of pathogen arthropods and providing alternative drugs with greater acceptance, especially in terms of environmental safety. Thus, the acaricidal activity showed in these assays by some natural monoterpenes sug-
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gests that some ofthese compounds might find use as alternative miticidal agents. EXPERIMENTAL TESTCOMPOUNDS.-AII monoterpenoids used in this study were purchased from Aldrich Chemical Company, Milan, Italy. TESTORGANISMS.-PSOroptic mites were isolated from naturally infested New Zealand white rabbits; the scabs and cerumen, collected from the infected ears, were observed by a stereoscopic microscope to identify the adult male and female mites of Pswopres cuniculi used for testing in this work. A voucher specimen (No. 95) has been deposited at the Insect Collection of the Dipartimento di Patologia Animale Profilassi ed Igiene degli Alimenti in Pisa.
BIOLOGICAL TESTING.-TWO different procedures were used to test the monoterpenes, direct contact and inhalation. In the former, dilutions of 1.0, 0.25, and 0.125% in physiological saline containing 20% vaseline oil were used. For each dilution,adoseofO.S ml wasplaced ina6-cmpetri dish containing 10 mites. Mites were placed in empty plates, in plates containing 0.5 ml ofphysiological saline, and in plates containing physiological saline plus 20% vaseline oil. Plates containing Acacerulen" (Teknofarma) were used as controls. Six replications for each dilution were made. All plates were placed at room temperature (22') and 70% relative humidity. All the motionless mites, observed by a stereoscopic microscope after a period of 48 h, were stimulated with a needle. Lack of any reaction and persistent immobility was presumed to indicate death (15). In the experiments carried out to test the acaricidal activity by inhalation of monoterpenes, mites(l0isample)wereplacedin 6-cmpetridishes covered with a filter paper disk which allowed gas exchange: each plate so prepared was inserted in a 9-crn petri dish containing the test substance and closed with the normal cover. Mites were exposed to dosages of 6, 3, and 1 p,I of each compound, measured with a Gilson P2 microtiter pipette (standard errork0.027 PI). At each dosage, 60 mites were tested. All plates were tested at rmm temperature (22') and 70% relative humidity. In these experiments all compounds used were pure, except for thymol and menthol which, as solids, were dissolved (1:6, wiw) in vaseline oil and, therefore, were tested at dosages six times higher (36,18, and 6 ~ 1 )The . control mites were exposed to the action of Acacerulen" (Teknofarma), physiological saline, and vaseline oil, all at 0.5 ml, and to the action of no substance (untested controls). All the motionless mites, observed by stereoscope microscope after a period of 24 h, were transferred into fresh 6-cm petri dishes and observed again
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Journal of Natural Products
h e r 24 h. Their mortality was evaluated as indicated before. T h e results are expressed as mean lethality percentagekstandard deviation of six replications.
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3.
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Reseived 29 December 1994
